The real-time polymerase chain reaction (PCR) is currently used as a diagnostic tool in clinical applications. The real-time PCR assay is carried out in the closed-tube format and can be used to obtain quantitative results. The chemistry of real-time PCR is based on monitoring fluorescence at every cycle at a set temperature that facilitates calculating the kinetics of the product formed and performing melting curve analysis to identify formation of the specific product. Fluorescence is usually monitored using an optical device to collect the data at specific excitation and emission wavelengths for the particular fluorescent dye present in the sample.
One method used to monitor nucleic acid amplification is the addition of SYBR Green I dye (Ririe et al., Anal. Biochem. 245:154-60, 1997) and LCGreen (Wittwer et al., Clin. Chem. 49:853-60, 2003) during PCR. During amplification, these fluorophores are excited with the appropriate wavelength of light, inducing fluorescence when the dye intercalates into a DNA double helix. However, this method lacks specificity and the primer-dimer can also fluoresce.
Specificity can be increased by using a labeled sequence-specific probe. Several of such methods are currently available for performing real-time PCR, such as TaqMan® probes (Lee et al., Nucleic Acids Res. 21:3761-6, 1993); molecular beacons (Tyagi and Kramer, Nat. Biotechnol. 14:303-8, 1996); self-probing amplicons (scorpions) (Whitcombe et al., Nat. Biotechnol. 17:804-7, 1999); Amplisensor (Chen et al., Appl. Environ. Microbiol. 64:4210-6, 1998); Amplifluor (Nazarenko et al., Nucleic Acids Res. 25:2516-21, 1997 and U.S. Pat. No. 6,117,635); displacement hybridization probes (Li et al., Nucleic Acids Res. 30:E5, 2002); DzyNA-PCR (Todd et al., Clin. Chem. 46:625-30, 2000); fluorescent restriction enzyme detection (Cairns et al. Biochem. Biophys. Res. Commun. 318:684-90, 2004); and adjacent hybridization probes (Wittwer et al., Biotechniques 22:130-1, 134-8, 1997).
Currently available labeled primers can have a secondary structure that is complex and in some instances must be synthesized using specialized procedures. For example LUX™ primers (Invitrogen Corp.) are fluorescently labeled on the 3′-end and have a stem-loop structure that must be denatured for the primer to work efficiently (especially for reverse transcription). The design of the LUX™ primer is also a time-consuming step, which requires specific software.
Several publications disclose probes that contain only one fluorophore for use in detecting the presence of a particular nucleic acid (for example see U.S. Pat. No. 6,699,661; U.S. Pat. No. 6,495,326; and U.S. Pat. No. 6,492,121 (all to Kurane et al.); U.S. Pat. No. 6,635,427 (Wittwer et al.); Kurata et al. (Nucl. Acids Res. 29:e34, 2001); Torimura et al. (Analyt. Sci. 17:155-60, 2001); and Crockett et al. (Analyt. Biochem. 290:89-97, 2001)). In these examples, the fluorophore is present on the very end of the probe and the fluorescent signal is either enhanced or quenched in the presence of the target nucleic acid sequence, depending on the particular design of the probe. In most cases, the labeled primer specifically hybridizes to the target nucleic acid sequence. Guo and Milewicz (Biotech. Lett. 25:2079-83, 2003) disclose universal fluorescent tag primers labeled on the 5′ end that are not sequence specific. The labeled fluorescent tag universal primer, in combination with two sequence-specific primers, are use to amplify a target nucleic acid sequence.
Yamane (Nucl. Acids Res. 30:e97, 2002) discloses a MagniProbe that has an internal fluorophore and an internal intercalator. The fluorescence is quenched by the intercalator in the absence of a target sequence. Upon hybridization with the target sequence, the probe emits fluorescence due to the interference in quenching by intercalation.
Nazarenko et al. (Nucl. Acids Res. 30:e37, 2002) disclose a probe with a single fluorophore near the 3′ end (but no quencher), and addition of 5-7 base pairs to the 5′ end of the sequence-specific probe, wherein the signal from the fluorophore is increased in the presence of the target sequence.